- Volume 64, Issue 7, 1983

Escherichia coli plasmids carrying a DNA restriction fragment corresponding to the extreme right end of the T7 bacteriophage genome render cells incompetent to support reproduction of wild-type T7. Processing of intracellular concatemeric phage DNA and cell lysis are perturbed by one such plasmid, pRS148. The results are discussed with reference to the possibility that the right end of the T7 genome encodes a lysis-related function.

The infectivity of Lactobacillus lactis bacteriophage LL-H was shown to be calcium-dependent. Of 10 different divalent cations screened, cadmium specifically decreased the infectivity of LL-H in the presence of calcium. At 1 to 2 mm, CdCl2 resulted in a decrease of the burst size of about 2.5- to 4-fold. Cd2+ was shown to reduce specifically the level of total phage DNA synthesis, resulting in a reduced progeny phage yield. Moreover, Cd2+ had the most profound irreversible effect on progeny phage production between 20 and 60 min after LL-H infection. This paralleled the beginning of phage DNA synthesis. Possible modes of action of Cd2+ on phage DNA replication are discussed.

Six sheep persistently infected with visna virus were studied for 4 1/2 to 5 3/4 years until they became ill. Virus was isolated at intervals from peripheral blood leukocytes and cerebrospinal fluid (CSF), and at the time of sacrifice from various parts of the brain and the lungs. Both brain and lungs showed lesions typical of advanced visna/maedi. All the sheep formed antibodies in sera and CSF. Virus isolates from each sheep were tested in neutralization tests against sera and CSF collected from the same animal. In one sheep all isolates were found to be identical to the inoculated virus by this test. In each of the other sheep an antigenic variant emerged from 1 to 3 years after inoculation and remained in circulation even after the formation of autologous antibodies. In one case a variant was isolated from the lungs, whereas in all cases the virus isolated from the brain was identical to the inoculated virus. The results show that antigenic variants are rare in visna and do not seem to have a role in the pathogenesis of the disease.

A group of 20 Icelandic sheep were infected intracerebrally with visna virus strain 1514, and 209 virus isolates were obtained from the blood, cerebrospinal fluid, and central nervous system (CNS) over a period of 7 years, during which eight animals developed clinical signs of visna necessitating sacrifice. (i) Using type-specific antisera, it was found that 12 (16%) of 76 isolates tested escaped neutralization. These 12 variant viruses were distributed randomly among animals and over time, and did not replace the infecting strain even though all sheep developed homotypic antibody within 3 months of infection. The one exception was sheep no. 1557 (an animal without clinical visna), where the last six isolates were variants. (ii) A total of 35 blood and CNS isolates from seven of these sheep (including five with clinical visna) were tested against serial samples of their own sera. Autologous antisera neutralized all isolates tested with the exception of isolates from sheep 1557. None of the isolates obtained at sacrifice from the five sheep with clinical visna escaped neutralization with autologous antisera. These data suggest that although variant viruses are encountered at considerable frequency during long-term infection of Icelandic sheep, the variants usually do not replace the infecting strain. Antigenic drift does not appear to be essential for virus persistence or for the development of clinically evident CNS lesions.

Dengue virus-infected mice showed a depressed antibody response to polyvinylpyrrolidone (PVP) when compared to controls. In both control and dengue virus-infected animals which were treated with anti-thymocyte serum (ATS) and primed with PVP, there was a heightened antibody response to PVP, suggesting that the anti-PVP response was controlled by T suppressor cells. The increase in the anti-PVP response in dengue virus-infected, ATS-treated animals was found to be similar to that seen in ATS-treated controls. T cells from infected animals could transfer suppression of anti-PVP response to normal mice, whereas the T cells from control animals could not induce significant suppression. The T cells from dengue virus-infected animals which had received 2,4-dinitrofluorobenzene (DNFB) tolerogen could induce in normal mice a significantly higher percentage of tolerance to contact sensitivity to DNFB when compared to the control T cells. The adherent and B cells from both infected and control animals failed to induce significant tolerance. These findings suggested that during dengue virus infection, there is enhanced T suppressor cell activity regulating the B cell response to PVP and T cell response to DNFB.

BHK-21 cells showed an increased ability to concentrate 2-deoxy-d-glucose (dGlc) 2 to 3 h after infection with vesicular stomatitis virus (VSV) or Semliki Forest virus (SFV), which began to be released at 2 and 3 h post-infection respectively; uptake of other nutrients was not affected in this way. Intracellular Na+ was either unchanged (VSV-infected cells) or increased (SFV-infected cells); K+ content was unchanged. These results do not support the current hypothesis that a non-specific increase in membrane permeability occurs in cells infected with rhabdoviruses or togaviruses.

Mumps virus infection of the CV-1 cell line results either in no cytopathic effect or extensive cell fusion, depending upon the infecting mumps virus strain. Growth cycle analyses indicated that both types of infection were the result of multiple cycle replication of mumps virus. Intracellular virus-specific polypeptide synthesis was examined by pulse- and pulse-chase-labelling with radioactive amino acids and sugars. The major polypeptides seen on SDS-polyacrylamide gels were NP (69000 mol. wt.), P (45 000 mol. wt.) and M (40000 mol. wt.); a non-structural polypeptide (22000 mol. wt.) was also present in infected cell lysates. The HN (74000 to 79000 mol. wt.) glycopolypeptide was detected in [3H]glucosamine- and [3H]mannose-labelled infected cells. A 65000 mol. wt. species that had incorporated these precursors was seen in pulse-labelled infected cell lysates, and this glycopolypeptide vanished during the chase interval with the concomitant appearance of two glycopolypeptides (59000 mol. wt. and 14000 to 15000 mol. wt.) which represented the F1 and F2 subunits of the F glycoprotein. Immunological data confirmed the relatedness of the 65000 mol. wt. glycopolypeptide to the F glycoprotein and identified it as the precursor F0. The F0 precursor glycopolypeptide was seen in cells infected with both fusing and non-fusing strains, and F0 was processed completely to F glycoprotein for all infections. Thus, the lack of cell fusion after infection with certain mumps strains is not the consequence of incomplete processing of the F0 precursor.

Temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were isolated and studied for interferon (IFN) induction in primary chick embryo (CE) cells. At the non-permissive temperature (41 °C), there was no viral RNA synthesis or IFN induction by u.v.-treated virions except for ts-3 (RNA+), which did synthesize RNA at 41 °C, and whose u.v.-treated virions did induce IFN at this temperature. Another mutant (ts-4) induced IFN without irradiation, at the permissive temperature (37 °C). The minimum u.v. target size for IFN inducibility was unaffected by the mutation and corresponded to about 5% of the genome required for the expression of infectivity. These results support the hypothesis that the appearance of NDV RNA immediately after infection (primary transcription) plays a key role in IFN induction.

A peptide, prepared by gentle oxidative detoxification of a methionine-free cobra α-neurotoxin, was evaluated for antiviral activities in vivo and in vitro. When added to BHK cells before infection the peptide reduced herpes simplex virus type 1 (HSV-1)-induced TCD50 in a dose- and time-dependent manner. The antiviral effect was also expressed on initiation of cell treatment 1 h after infection if the peptide was left in contact with cells. Single, intracerebral doses of the toxoid in suckling mice significantly increased 50% survival times, and reduced virus replication in the infected brain tissues. Cutaneous lesions and resultant scar formation induced by HSV-1 in hairless mice were also significantly reduced by subcutaneous peptide injections.

By several criteria, replication of herpes simplex virus type 1 (HSV-1) in primary cell cultures from inbred strains of mice resistant (R) to this virus was less than in cultures from susceptible (S) strains. The difference was not obviously related to less efficient adsorption, to more efficient production of interferon or to a larger number of lysosomes. Except at high multiplicities of infection, cells from the F1 progeny of a cross between an R and an S strain replicated virus as well as cells from the S parent; this contrasts with strain-related resistance to intraperitoneal inoculation in vivo, which is inherited as a dominant characteristic. It is suggested that diminished ability of structural cells to replicate HSV-1 may contribute to resistance in the intact animal.

The originally infected ear of mice latently infected in the cervical ganglia with herpes simplex virus (HSV) was treated with one of five stimuli: stripping with cellophane tape, irradiation with u.v. light, or the application of xylene, dimethyl sulphoxide (DMSO) or retinoic acid. Each of these stimuli induced the appearance of infectious virus in the ganglia 1 to 5 days later, most frequently after 1 to 3 days. Virus was also isolated from the treated ears, most frequently 3 to 5 days after stimulation. In a proportion of mice treated with cellophane tape stripping, xylene, retinoic acid or DMSO, clinical recurrent disease was observed, although in the case of DMSO this proportion was low. Some of the physiological changes induced in the skin by the five stimuli were studied. Treatment with DMSO, cellophane tape stripping or xylene induced almost immediate inflammation in the skin as judged by extravasation of Evans blue dye. Studies with inhibitors suggested that this was mediated by a neurogenic factor together with histamine or 5-hydroxytryptamine, or both of these. In addition, with the exception of mice treated with DMSO, the levels of prostaglandins of the E and F classes in the skin of the ear were elevated 1 to 3 days after treatment. These results are discussed with reference to the mechanisms by which recurrent herpetic disease develops.

The synthesis of virus polypeptides in rat XC cells infected with herpes simplex virus type 1 (HSV-1; 13VB4tsC75) was studied. At the permissive temperature the virus induced the synthesis, in a cascade fashion, of significant amounts of several early polypeptides (ICP 6, 8 and 39) and those late polypeptides that are relatively resistant to inhibition by phosphonoacetic acid in HEp2 cells (ICP 5, 11, 25, 29, 43 and 44). The infectious cycle appeared to become arrested in XC cells at about 7 to 9 h post-infection, because the relative concentrations of early and latest polypeptides labelled thereafter remained constant and the levels of several of the late virus polypeptides were severely reduced (ICP 2, 10, 24 and 26) or not synthesized at all (ICP 32, 34 and 37). When XC cells were infected at a very high m.o.i., only a small amount of virus DNA synthesis could be detected; the synthesis of cellular DNA was not impaired and the infected XC cells continued to replicate for several weeks at least. When XC cells were infected at the non-permissive temperature, only the immediate-early (IE) ICP 4 could be detected while IE ICP 0 and 22 were not observed. Infection of XC cells with HSV-1 (MP) also resulted in the production of early and late viral polypeptides. On the other hand, in XC cells infected with HSV-1 (F) and HSV-1 (HFEM), the synthesis of virus polypeptides could not be detected.

A human papillomavirus type 1 (HPV-1)/pBR322 recombinant plasmid was constructed consisting of two complete HPV-1 genomes in a head-to-tail arrangement, inserted into the BamHI site of pBR322. To obtain established human keratinocytes carrying dimeric HPV-1, origin-minus simian virus 40 (SV40) DNA was used as a dominant transforming marker in co-transfection experiments performed with cultured human foetal keratinocytes. Using the Southern blotting technique, HPV-1 DNA was detected in one out of five SV40-transformed human keratinocyte cell lines which were obtained in this way. Further blotting experiments using SV40, pBR322 and HPV-1 DNA as probes revealed patterns of hybridization which were consistent with co-integration of SV40 DNA with between two and four copies of the HPV-1 genome. The HPV-1 sequences in this cell line were virtually non-methylated and were transcribed at a lower level than SV40 mRNAs in the same cultured cells. A low level of monomeric HPV form I DNA was detected by blotting of undigested total cellular DNA. No evidence for a stimulation of form I HPV DNA replication could be obtained by blotting analysis of total DNA extracted from keratinizing cysts, which were formed by subcutaneous inoculation of these cells into nude mice.

The rabbit-adapted Borna disease (BD) virus strain V was passaged by intracerebral infection of 1-day-old Wistar rats. Infectivity titres reached 108 infectious units per gram of brain 4 weeks after infection. No clinical signs were evident. The persistent infection could be induced with adapted or field strains of BD virus. Strains were identified by neutralization tests. The virulence of the rabbit-adapted BD virus for the rat increased with rat passages. The 5th passage induced clinical symptoms in animals infected at 1 week of age or older. Between 20% and 50% of diseased rats died. Virus-specific antigen was detectable immunohistologically in neurons of rats infected at all ages. Animals inoculated at 1 or 2 months of age, but not the neonatal rats, showed signs of inflammation in the brain. Infected rats produced specific antibodies. In the older groups (infected at ages of 1 or 2 months), and especially in surviving animals, occasionally, neutralizing antibodies with high titres were found. Transfer of primed spleen cells resulted in subacute disease. These findings demonstrate that neonatal rats can acquire a persistent, tolerant infection and that expression of disease is mediated by immunological factors.

A mutant (m-29) of Autographa californica nuclear polyhedrosis virus (AcMNPV) grew in Spodoptera frugiperda and Trichoplusia ni cells but did not form typical intranuclear occlusion bodies (OB); instead, small particles (95 to 180 nm diam.) were produced in copious amounts within nuclei. Ultrastructural studies showed that the particles did not occlude enveloped nucleocapsids and that they lacked a macromolecular paracrystalline lattice and a structure equivalent to the occlusion body envelope. The particles within nuclei stained in an immunofluorescence test with anti-polyhedrin antibody and when extracted from cells the major polypeptide of a particle preparation was indistinguishable from polyhedrin when examined on SDS-polyacrylamide gels and had an identical peptide pattern following proteolysis with V8 protease. Other elements believed to be implicated in OB morphogenesis such as a proliferation of intranuclear membranes, enveloped bundles of nucleocapsids, patches of fibrous material and fibrous sheets were present in normal amounts. No alteration in the synthesis or processing of polypeptides was seen in mutant-infected cells. Analysis of m-29 DNA with BamHI, EcoRI and HindIII restriction endonucleases revealed that the HindIII restriction site at the F/V junction of viral DNA was absent in the mutant. No other modifications in the restriction patterns were detected. It is proposed that an alteration in the amino acid sequence of polyhedrin towards the — NH2 terminus of the polypeptide may account for the growth characteristics of the mutant.

Two distinct antiviral activities can be detected in L cells treated with low levels of interferon and infected with a one-step interferon-sensitive mutant of mengovirus (is-1). The first antiviral activity (AVA-1) primarily delayed virus RNA and protein synthesis and thereby lengthened the virus replication cycle. It did not prevent cell death. The second antiviral activity (AVA-2) allowed the virus-induced inhibition of host macromolecular synthesis but inhibited all other virus functions. By 9 to 12 h post-infection host synthesis resumed and most cells survived. The data suggest that some step in the virus replication cycle activates AVA-2 leading to the destruction of the virus genome 6 to 12 h after infection. In unprotected cells the yields of parental virus (is +) and is-1 were similar. No qualitative or quantitative differences in virus products were observed by several techniques. The is-1 virus seems to have lost a wild-type function which normally blocks the action of AVA-2.

Protein subunit vaccines were prepared from a mixture of the haemagglutinin (HN) and fusion (F) glycoproteins of parainfluenza type 3 virus (PI-3). The glycoproteins were isolated in three different forms and characterized by their sedimentation coefficients: 30S protein micelles (a complex of several HN and F glycoproteins devoid of detergent and lipid), 18S protein-TX complexes (a complex of several glycoproteins containing the detergent Triton X-100), and 4S protein-TX complexes (probably monomers of the glycoproteins complexed to Triton X-100). These preparations were tested as vaccines in mice and lambs. The immune response in the mice was assayed both in the serum and in extracts from the lungs using an ELISA technique. Both of the multimeric complexes were highly immunogenic. The 30S protein micelles induced a high antibody response after two injections with either 10 or 1 µg protein. The serum IgG titres reached levels of about 90 µg/ml and 40 µg/ml respectively. Similar titres were reached with the 18S protein-TX complexes. After two injections of either the 30S or the 18S complexes IgA antibody responses were detected in the lung extracts. The 4S protein-TX complexes were poor immunogens and induced low antibody responses in mice. The lambs were vaccinated with the 30S protein micelles, and the immune response was evaluated serologically and in challenge experiments. The 30S protein micelles in an oil adjuvant induced detectable serum antibody titres as well as protective immunity against the pneumonia caused by the PI-3 virus.

The Onderstepoort strain of canine distemper virus (CDV) which has been adapted to newborn Swiss mice was used in a study in weanling mice. Intracerebral inoculation of newborn mice with either the parent or mouse-adapted virus strain led to mortality in 100% of the animals. In weanling mice the parent virus produced less than 10% mortality, whereas the mouse-adapted strain killed approximately 40% of the animals and this was not dose-dependent. Although a meningitis was observed in the surviving mice, the occurrence of viral antigens was less widespread in the brain of weanling mice than in the brain of newborn mice, and could not be detected more than 5 months after infection. In long-term experiments two phenomena were observed. At 4 to 6 months post-infection up to 30% of the mice became obese; however, no viral antigens were detected in the brains. At 13 to 17 months post-infection a number of mice became paralysed and virus antigen could be detected in the brain and lymph glands; however, infectious virus could not be isolated. These observations are discussed in relation to neurological infection and metabolic disease.

Heterotypic complementation between temperature-sensitive (ts) mutants of Sindbis (SIN) and Western equine encephalitis (WEE) viruses occurs under appropriate conditions. One heterotypic pair, SIN ts153 × WEE ts39 showed efficient complementation, and four other combinations gave detectable complementation, indicating that these two viruses, which are closely related serologically and biochemically, are sufficiently closely related to complement each other functionally. Cells mixedly infected with ts mutants or wild-type strains of both SIN and WEE viruses produced phenotypically mixed virions, in addition to both parental viruses. Various types of phenotypically mixed virions have been identified by neutralization with corresponding antisera, by thermal inactivation and by temperature sensitivity of replication. Some virions contained WEE genomes and envelopes containing primarily SIN proteins. Other phenotypically mixed virus yields contained primarily doubly neutralizable viruses which are presumed to have a mosaic of envelope proteins. Phenotypically mixed virions were morphologically indistinguishable from the parental types.

Herpes simplex virus (HSV) types 1 and 2 reactivate from dissociated cultured dorsal root ganglia of latently infected mice. The neurons in culture were identified morphologically and by using the specific anti-neuronal monoclonal antibody A2B5. HSV antigen expression during reactivation was first seen in neurons on day 3 after dissociation, and infectious virus was released subsequently. Approximately 0.4% of neurons released infectious virus. The presence of neutralizing antibody to HSV did not modify the reactivation process. After infection of mouse neuronal cultures in vitro with HSV, cytopathic effect and viral antigen expression appeared within 18 h predominantly in fibroblastic cells.